U.S. patent number 7,802,517 [Application Number 11/477,655] was granted by the patent office on 2010-09-28 for method of patterning molecules on a substrate using a micro-contact printing process.
This patent grant is currently assigned to Sony Deutschland GmbH. Invention is credited to Gregor Kron, Dirk Mayer, Andreas Offenhaeusser, Daniel Schwaab, Jurina Wessels, Akio Yasuda.
United States Patent |
7,802,517 |
Wessels , et al. |
September 28, 2010 |
Method of patterning molecules on a substrate using a micro-contact
printing process
Abstract
The present invention relates to a method of patterning
molecules on a substrate using a micro-contact printing process, to
a substrate produced by said method and to uses of said substrate.
It also relates to a device for performing the method according to
the present invention.
Inventors: |
Wessels; Jurina (Stuttgart,
DE), Kron; Gregor (Stuttgart, DE), Yasuda;
Akio (Esslingen, DE), Schwaab; Daniel (Wesseling,
DE), Mayer; Dirk (Frechen, DE),
Offenhaeusser; Andreas (Eynatten, BE) |
Assignee: |
Sony Deutschland GmbH (Cologne,
DE)
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Family
ID: |
36096436 |
Appl.
No.: |
11/477,655 |
Filed: |
June 30, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070098899 A1 |
May 3, 2007 |
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Foreign Application Priority Data
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Nov 2, 2005 [EP] |
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05023880 |
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Current U.S.
Class: |
101/483; 977/793;
977/789; 977/887 |
Current CPC
Class: |
B05C
1/027 (20130101); G03F 7/0002 (20130101); B81C
1/00206 (20130101); B82Y 10/00 (20130101); B41M
3/006 (20130101); B82Y 40/00 (20130101); Y10S
977/793 (20130101); Y10S 977/887 (20130101); Y10T
428/24926 (20150115); B01J 2219/00626 (20130101); B01L
3/0258 (20130101); B01J 2219/00637 (20130101); B01J
2219/0063 (20130101); B01J 2219/0061 (20130101); B01J
2219/00725 (20130101); Y10T 428/24802 (20150115); B01J
2219/00382 (20130101); B01J 2219/00612 (20130101); B01J
2219/00659 (20130101); Y10S 977/789 (20130101); B01J
2219/00527 (20130101); Y10T 428/24917 (20150115); B01J
2219/00605 (20130101) |
Current International
Class: |
B41M
3/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 2004/022338 |
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Mar 2004 |
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WO |
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WO 2005/058478 |
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Jun 2005 |
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WO |
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Other References
Hovis, J.S. and S.G. Boxer. Patterning barriers to lateral
diffusion in supported lipid bilayer membranes by blotting and
stamping. Langmuir 16:894-897; 2000. cited by examiner .
Tseng et al. Protein micro arrays immobilized by .mu.-stamps and
-protein wells on PhastGel1 pad. Sensors and Actuators B 83:22-29
(2002). cited by examiner .
Tan et al. Microcontact printing of proteins on mixed
self-assembled monolayers. Langmuir 18:519-523 (2002). cited by
examiner .
GenBank GI:162648 [online] Feb. 11, 2002 [retrieved on Mar. 10,
2009] retrieved from http://www.ncbi.nlm.nih.gov/protein/162648.
cited by examiner .
Libioulle et al. Contact-inking stamps for microcontact printing of
alkanethiols on gold. Langmuir 15:300-4, 1999. cited by examiner
.
Delamarche, E. "Microcontact Printing of Proteins" in
Nanobiotechnology, 2004, Wiley-VCH Verlag GmbH & Co. KgaA,
Weinheim, pp. 31-52. cited by examiner .
Oliva et al. Patterning axonal guidance molecules using a novel
strategy for microcontact printing. Neurochemical Research
28(11):1639-1648, Nov. 2003. cited by examiner .
Zhou et al. Reversible hydrophobic barriers introduced by
microcontact printing: application to protein microarrays.
Microchim. Acta 146, 193-205 (2004). cited by examiner.
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Primary Examiner: Woolwine; Samuel
Attorney, Agent or Firm: Oblon, Spivak, McClelland, Maier
& Neustadt, L.L.P.
Claims
The invention claimed is:
1. A method of patterning molecules on a substrate using a
micro-contact printing process, whereby the molecules to be
patterned are kept in solvent or are covered by solvent during the
entire micro-contact printing process, said method comprising:
providing molecules to be patterned in a solvent and providing a
patterned surface, said molecules being first immobilized on an
ink-pad within said solvent; transferring said molecules to be
patterned onto said patterned surface and immobilizing them thereon
while keeping said molecules in said solvent or covered by said
solvent, said ink-pad having said molecules immobilized thereon is
brought into conformal contact with said patterned surface in a
first solvent environment containing said molecules to be patterned
and said solvent, thereby transferring said molecules onto said
patterned surface and immobilizing them thereon; and providing a
substrate in a second solvent environment, transferring said
patterned surface having said molecules immobilized thereon to said
second solvent environment, and bringing said patterned surface
having said molecules immobilized thereon into conformal contact
with said substrate, thereby creating a pattern of said molecules
on said substrate, while keeping said molecules in said solvent or
covered by said solvent, wherein said molecules to be patterned are
protein molecules, wherein said micro-contact printing process
occurs in the absence of a drying step.
2. The method according to claim 1, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 180
min after immobilizing said molecules to be patterned on said
patterned surface.
3. The method according to claim 1, wherein, after the providing a
substrate, said patterned surface is lifted from said substrate,
thereby leaving behind a substrate having a pattern of said
molecules thereon.
4. The method according to claim 3, wherein said substrate having a
pattern of molecules thereon is kept in or covered by solvent
containing a buffer.
5. The method according to claim 1, wherein said molecules to be
patterned retain their function and/or activity and/or native
conformation throughout the entire process, due to their being kept
in solvent or covered by solvent during the entire micro-contact
printing process.
6. The method according to claim 1, wherein said substrate
comprises a spacer layer and/or a binding layer which facilitates
binding of said substrate to said molecules to be patterned through
covalent binding, electrostatic forces, van der Waals forces,
H-bonding, London forces or any combination of the foregoing.
7. The method according to claim 1, wherein said substrate is
selected from the group consisting of: metals and semi metals,
single or polycrystalline materials; single or polycrystalline
metals and semi metals, gold, platinum, or silicon, or; composite
materials of single or polycrystalline composites, siliconoxide, or
GaAs, or amorphous composite materials or glass; plastics, or
elastomers or polydimethylsiloxane, or plastomers, or polyolefines,
or ionomers, or resist materials, or UV-NIL resists.
8. The method according to claim 1, wherein said patterned surface
is made from a material selected from the group consisting of:
single-crystalline materials and polycrystalline materials,
silicon, silicon oxide, layered composite systems, silicon oxide on
silicon, metal layers on silicon or metal layers on silicon oxide;
amorphous materials, glass; plastics, elastomers,
polydimethylsiloxane, plastomers, polyolefines (POP, polyolefinic
plastomers), ionomers, resist materials, or UV-NIL-resists.
9. The method according to claim 1, wherein at least a surface of
said ink-padis made from a material selected from the group
consisting of single-crystalline materials and polycrystalline
materials, silicon, silicon oxide, layered composite systems,
silicon oxide on silicon, metal layers on silicon/siliconoxide;
amorphous materials, glass; plastics, elastomers,
polydimethylsiloxane, plastomers, polyolefines (POP, polyolefinic
plastomers), ionomers, resist materials, or UV-NIL-resists.
10. The method according to claim 1, wherein said molecules to be
patterned are selected from the group consisting of: redox
proteins, nucleic-acid binding proteins, metallo-proteins,
cytochrome c, azurin, cytoskeleton-proteins.
11. The method according to claim 1, wherein said protein molecules
to be patterned have one or plural lysine residues, and wherein
said substrate is Au, or Au having a spacer layer on its surface so
as to avoid denaturation of said protein, said spacer layer having
a thickness in the range of from 0.5 nm to 200 nm.
12. The method according to claim 1, wherein the pattern comprises
features having a length in the range of from approximately 10 nm
to 500 .mu.m.
13. The method according to claim 1, wherein said providing a
patterned surface includes providing a patterned surface in the
form of a stamp.
14. The method according to claim 1, wherein said providing
molecules to be patterned in a solvent includes providing molecules
first immobilized on an ink-pad in the form of a non-patterned
surface within said solvent.
15. The method according to claim 1, wherein said first solvent
environment also includes a buffer.
16. The method according to claim 1, wherein said second solvent
environment also includes a buffer.
17. The method according to claim 1, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 120
min after immobilizing said molecules to be patterned on said
patterned surface.
18. The method according to claim 1, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 10 min
after immobilizing said molecules to be patterned on said patterned
surface.
19. The method according to claim 1, wherein the bringing into
conformal contact of said patterned surface with said substrate of
the providing a substrate occurs in a period not longer than 1 min
after immobilizing said molecules to be patterned on said patterned
surface.
20. The method according to claim 1, wherein the pattern comprises
features having a length in the range of from approximately 10 nm
to .gtoreq.200 nm.
21. The method according to claim 1, wherein the pattern comprises
features having a length in the range of from approximately 10 nm
to .gtoreq.150 nm.
22. The method according to claim 1, wherein the substrate is
modified with a molecular layer.
23. The method of claim 22, wherein the molecular layer is a self
assembling monolayer (SAM).
24. The method of claim 23, wherein the molecules of the SAM have
two termini, a first terminus for binding to the substrate, and a
second terminus for binding the protein molecules to be
patterned.
25. The method of claim 24, wherein the substrate comprises gold
and the first terminus comprises a thiol for binding to the
gold.
26. The method of claim 24, wherein the substrate comprises silicon
oxide and the first terminus comprises a silane for binding to the
silicon oxide.
27. The method according to claim 24, wherein the SAM is selected
from the group consisting of: a SAM with mercapto- or amino-groups
for binding metals, a SAM with carboxy-groups for electrostatic
binding, a SAM with mercapto-groups for binding metals, with plain
alkyl chains having methylene groups for van der Waals interaction,
with --COOH, --OH or vinyl-groups for covalent coupling, or SAMs
with antibodies for binding corresponding antigens, or SAMs with
antigens for binding corresponding antibodies, or SAMs with
receptors for specific binding of molecules.
28. The method according to claim 22, wherein the molecular layer
includes antibodies for binding antigens or includes antigens for
binding corresponding antibodies.
29. The method according to claim 22, wherein the molecular layer
includes receptors for specific binding of molecules.
30. The method according to claim 1, wherein the substrate is gold
modified with a mercapto undecanoic acid layer (MUA).
Description
FIELD
The present invention relates to a method of patterning molecules
on a substrate using a micro-contact printing process, to a
substrate produced by said method and to uses of said substrate. It
also relates to a device for performing the method according to the
present invention.
BACKGROUND
During the past decade, soft lithography has developed to a
versatile technique for fabricating chemically micro- and
nanostructured surfaces [1,2]. Among several techniques known
collectively as soft lithography, micro-contact printing (.mu.CP)
has become the most commonly used method [1]. A patterned polymer
stamp is covered with an ink of molecules using either contact
inking or wet inking. In contact inking the solvent is reduced to
the dry state while the molecules self assemble on an inkpad. The
molecules are transferred onto the stamp under ambient conditions
by bringing the stamp and the inkpad into conformal contact. In the
wet inking process, the ink is poured over the stamp and then
reduced under a stream of nitrogen to a dry state. In both cases
the molecules are on the stamp prior to the transfer onto a
substrate. For the transfer of the ink onto the substrate, stamp
and substrate are brought into conformal contact with a substrate
for the transfer of the molecules from the stamp to the substrate
[3,4].
Recently, also proteins have been transferred to a variety of
substrates [5-7]. The advantage of .mu.CP thereby is the direct,
fast and gentle transfer of proteins, however, all
.mu.CP-techniques reported so far ultimately lead to a denaturation
of the printed proteins. Native proteins immobilized onto modified
surfaces are of major interest for sensor technology, cell
culturing and micro-biology. One application is e.g., the
patterning of growth factor proteins on silicon oxide for guiding
cell growth [8].
A critical issue for the immobilization of biomolecules, e.g.
proteins, nucleic acids etc. on surfaces is their denaturation and
hence the loss of the functionality after their immobilization. The
functionality, as e.g. in the case of cytochrome c (cyt c), may
depend on the orientation and conformation of the protein on the
surface. So far, the immobilization and redox activity of cyt c has
been investigated on chemically modified Au surfaces [9-11] and on
ITO [12]. Runge et al. reported a process for the transfer for cyt
C molecules onto ITO surfaces, in which the proteins are dried on
the stamp [12]. For ITO-surfaces it could be demonstrated that the
reactivity of the proteins depend on the surface modification of
the stamps used for the process [11].
In addition to transferring proteins, a method for
transfer-printing of highly aligned DNA nanowires has been
described by Nakao et al. [13] using PDMS stamps. In this method
hydrodynamic forces are used to align DNA on PDMS. After the
alignment step the PDMS stamp is brought into conformal contact
with a mica sheet for the transfer of DNA onto mica. AFM images
showed that the apparent height of the as transferred DNA is
between 0.27 and 0.35 nm, indicating that the DNA molecules are
probably elongated and possibly sheared as a result of the
hydrodynamic forces.
However, all the above described inking methods used in the prior
art cause denaturation of the protein(s) and loss of their
activity.
SUMMARY
Accordingly, it was an object of the present invention to provide
for a method allowing the immobilization and patterning of
molecules on a substrate, whereby the molecules to be patterned and
immobilized retain their function and/or native conformation and/or
activity. Furthermore, it was an object of the present invention to
provide for a method of patterning molecules on a substrate that is
easy to perform even with biological macromolecules whilst
maintaining their functionality. Furthermore, it was an object of
the present invention to provide for a method of patterning
molecules on a substrate whereby pattern features .ltoreq.200 nm
can be achieved.
All these objects are solved by a method of patterning molecules on
a substrate using a micro-contact printing process, whereby the
molecules to be patterned are kept in solvent or are covered by
solvent during the entire micro-contact printing process.
Such a micro-contact printing process in which the molecules to be
patterned are kept in solvent or are covered by solvent during the
entire process is herein also sometimes referred to as "in-situ
printing process" or "in-situ stamping process". The term "in-situ
printing process" or "in-situ stamping process" as used herein, is
meant to denote a micro-contact printing process whereby the
molecules to be patterned retain their functionality and/or
conformation as a result of being kept in solvent or being covered
by solvent during the entire printing process.
In a preferred embodiment such "in-situ printing process is meant
to denote a micro-contact printing process in the entire course of
which the molecules to be patterned are kept in their respective
physiological conditions that allow them to retain their native
functionality and/or conformation. It should be emphasized that the
term "physiological conditions" will depend on the type of
molecules to be patterned. For example, if the molecules to be
patterned are molecules of an oxygen transporting protein, the
"physiological conditions" for such a molecule will preferably
include a pH-value in the range of from 7.0 to 7.8, preferably
around pH 7.4. If, on the other hand, the molecules to be patterned
are molecules of a gastric enzyme, the "physiological conditions"
for such a molecule will include a pH-value in the range of from
1.8 to 4. Hence, overall the term "physiological conditions" will
include pH-values that may range from 1 to 10.
In one embodiment said micro-contact printing process occurs in the
absence of a drying step.
In one embodiment, the method according to the present invention
comprises the following steps: a) providing molecules to be
patterned in a solvent and providing a patterned surface,
preferably in the form of a stamp, b) transferring said molecules
to be patterned onto said patterned surface and immobilizing them
thereon whilst keeping said molecules in said solvent or covered by
said solvent, c) providing a substrate and bringing said patterned
surface having said molecules immobilized thereon into conformal
contact with said substrate, thereby creating a pattern of said
molecules on said substrate, whilst keeping said molecules in said
solvent or covered by said solvent.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: SEM image of cyt c on (MUA)/gold The lines are 1 .mu.m to
150 nm with equal gaps in between. The dark lines are cyt c
molecules.
FIG. 2: Cyclic Voltammograms (scan rate: 50 m V/s; reference
electrode: SCE) of cyt c on MUA/gold. Reference substrate without
cyt c (-) cyt c absorbed from solution (.tangle-solidup.), cyt c
after in-situ stamping or printing (.box-solid.), and cyt c after
ambient stamping or printing (.cndot.)
FIG. 3: Cyclic Voltammograms (scan rate: 50 m V/s; reference
electrode: SCE) of cyt c on MUA/gold. Comparison of different times
for which the stamp remains in a buffer reservoir before contacting
the substrate, namely 5 s (.box-solid.), 10 min (.tangle-solidup.)
and 2 h (-).
FIG. 4: shows a schematic representation of the various schemes 1-4
that are specific embodiments of the present invention.
DETAILED DESCRIPTION
The term "to bring into conformal contact with" is meant to denote
a contact between two entities, e.g. surfaces, allowing the
transfer of molecules that were on one entity before the contact,
to the other entity. In some embodiments, exertion of pressure is
needed for such transfer to occur, and in these instances, the term
"to bring into conformal contact with" is to be equated with "to
press on(to)".
The term "to immobilize a molecule on a surface", as used herein,
is meant to denote an activity by which a molecule becomes attached
to a surface, It does not mean that a molecule thus immobilized
will be unable to move completely. For example, parts of a molecule
thus immobilized may still rotate about certain chemical bonds
and/or may "swing" within the solvent covering the surface.
"Immobilization", as used herein, merely implies some kind of
attachment of a molecule to a surface which attachment prevents the
molecule to diffuse freely from said surface. In its simplest form,
such immobilization of molecules on a surface may occur by exposing
said surface to said molecules.
In one embodiment in step a), said molecules are provided in said
solvent and are first immobilized on an ink-pad, preferably in the
form of a non-patterned surface, within said solvent, wherein,
preferably, in step b), said ink-pad having said molecules
immobilized thereon is brought into conformal contact with said
patterned surface in a first solvent environment containing said
molecules to be patterned, said solvent and, optionally, a buffer,
thereby transferring said molecules onto said patterned surface and
immobilizing them thereon, and wherein, more preferably, in step
c), said substrate is provided in a second solvent environment
containing said solvent and, optionally a buffer, and wherein said
patterned surface having said molecules immobilized thereon, after
step b), is transferred to said second solvent environment and is
brought into conformal contact with said substrate. The first and
second and subsequent solvent environments contain a solvent and
may, in addition thereto, also contain a solute, such as a salt,
preferably a buffer, more preferably a buffer by the presence of
which physiological conditions are established or conditions are
established which mimic a physiological state. The second and
subsequent solvent environments initially contain no molecules to
be patterned or only a very small amount thereof. As soon as the
patterned surface or the ink-pad has been transferred to said
second, third, fourth etc. solvent environment, however, there will
be some molecules to be patterned present in said solvent
environment.
The term "ink-pad", as used herein, is meant to signify any surface
that is capable of acting as a transfer-facilitating surface for
molecules to be patterned. In its simplest form it may simply be a
non-patterned surface. Under certain conditions, however, it may
also be some sort of surface that has a pattern on it, and/or that
has the capacity of absorbing molecules to be patterned and the
capacity of releasing some of these molecules upon bringing a stamp
into conformal contact with said ink-pad.
In one embodiment in step b), said ink-pad having said molecules
immobilized thereon is brought into conformal contact with said
patterned surface in a second solvent environment containing said
solvent and, optionally, a buffer, after transfer of said ink-pad
having said molecules immobilized thereon to said second solvent
environment, thereby transferring said molecules onto said
patterned surface and immobilizing them thereon, wherein,
preferably, in step c) said substrate is provided in a third
solvent environment containing said solvent and, optionally, a
buffer, and wherein said patterned surface having said molecules
immobilized thereon, after step b), is transferred to said third
solvent environment and is brought into conformal contact with said
substrate.
In one embodiment in step a), said molecules are provided in said
solvent and, in step b), said molecules are immobilized on said
patterned surface within said solvent, wherein, preferably, step b)
occurs by immersing said patterned surface in said solvent, and
wherein, more preferably, in step c), said substrate is provided in
a fourth solvent environment containing said solvent and,
optionally, a buffer and wherein said patterned surface having said
molecules immobilized thereon, after step b), is transferred to
said fourth solvent environment and is brought into conformal
contact with said substrate.
In one embodiment in step c), said substrate is provided without a
solvent environment, and wherein said patterned surface having said
molecules immobilized thereon is brought into conformal contact
with said substrate whilst keeping said molecules covered by said
solvent, and wherein said patterned surface is transferred to a
fifth solvent environment containing said solvent and, optionally a
buffer, whilst being in contact with said substrate, said transfer
of said patterned surface and said substrate occurring immediately
after said patterned surface is brought into conformal contact with
said substrate, so as to avoid a drying of said patterned surface
on said substrate.
In one embodiment, said step b) is performed over a period in the
range of from 1 s to 60 min. Step b) may be considered an "inking
step".
Preferably, the bringing into conformal contact of said patterned
surface with said substrate of step c) occurs in a period not
longer than 180 min after immobilizing said molecules to be
patterned on said patterned surface, preferably not longer than 120
min, more preferably not longer than 10 min, most preferably not
longer than 1 min after immobilizing said molecules to be patterned
on said patterned surface.
As used in this context, the term "occurs in a period not longer
than . . . after immobilizing . . . " is meant to denote that said
bringing into conformal contact must take place within a period of
180 min at a maximum, said period commencing from the time that
said molecules to be patterned are immobilized on said patterned
surface.
In one embodiment after step c), said patterned surface is lifted
from said substrate, thereby leaving behind a substrate having a
pattern of said molecules thereon, wherein, preferably, said
substrate having a pattern of molecules thereon is kept in or
covered by solvent optionally containing a buffer.
In one embodiment said molecules to be patterned are selected from
the group comprising proteins, nucleic acids, preferably DNA or
RNA, lipids and combinations of any of the foregoing, wherein,
preferably, said molecules to be patterned are protein
molecules.
Preferably, said molecules to be patterned retain their function
and/or activity and/or native conformation throughout the entire
process, due to their being kept in solvent or covered by solvent
during the entire micro-contact printing process, wherein, more
preferably, said molecules to be patterned are kept under
physiological conditions, as measured by, for example, pH and
salinity, throughout the entire micro-contact printing process.
The term "solutes" as used in this context, does not exclude the
presence of solutes within the solvent. In fact, these may be
preferred in order to establish the desired physiological
conditions. Such solutes, without being limited thereto, include
salts and their ion-components, buffers, proteins, nucleic acids
and lipids.
In one embodiment said substrate has a hydrophilic surface if said
molecules to be patterned are hydrophilic, and wherein said
substrate has a hydrophobic surface if said molecules to be
patterned are hydrophobic.
Preferably, said substrate comprises a spacer layer and/or a
binding layer which facilitates binding of said substrate to said
molecules to be patterned through covalent binding, electrostatic
forces, van der Waals forces, H-bonding, London forces or any
combination of the foregoing.
In one embodiment said substrate is selected from the group
comprising metals and semi metals, single or polycrystalline
materials; preferably single or polycrystalline metals and semi
metals (most preferably gold, platinum, silicon) or; composite
materials preferably single or polycrystalline composites (most
preferably (siliconoxide, GaAs) or amorphous composite materials
(most preferably glass); plastics, preferably elastomers (most
preferably polydimethylsiloxane), preferably plastomers (most
preferably polyolefines), preferably ionomers, preferably resist
materials (most preferably UV-NIL resists); any of the afore
mentioned materials modified with molecular layers, preferably SAMs
(self assembling monolayers), for direct binding or indirect
binding, SAMs for indirect binding will be with one or multiple
chemicals or treatments to achieve the desired binding site; most
preferably SAMs with two termini: one for attaching the molecule to
the substrate such as a thiol-headgroup for binding on gold; most
preferably SAMs with a silane-headgroup terminus for binding on
siliconoxide; the second terminus for coupling the ink, such as
SAMs with mercapto- or amino-groups for binding metals, SAMs with
carboxy-groups for electrostatic binding, most preferably SAMs with
mercapto-groups for binding metals, with plain alkylchains having
methylene groups for van der Waals interaction, with --COOH, --OH
or vinyl-groups for covalent coupling; or SAMs with antibodies for
binding corresponding antigens, or SAMs with antigens for binding
corresponding antibodies, or SAMs with receptors for specific
binding of molecules; or any of the aforementioned materials
modified with molecular layers, with antibodies for binding
corresponding antigens, or modified with molecular layers with
antigens for binding corresponding antibodies, or modified with
molecular layers with receptors for specific binding of molecules;
most preferably gold modified with a mercapto undecanoic acid layer
(MUA).
Preferably, said patterned surface is made from a material selected
from the group comprising single-crystalline materials and
polycrystalline materials, such as silicon, silicon oxide, layered
composite systems, such as silicon oxide on silicon, metal layers
on silicon/silicon oxide; amorphous materials, such as glass;
plastics, such as elastomers, preferably polydimethylsiloxane,
plastomers, preferably polyolefines (POP, polyolefinic plastomers),
ionomers, resist materials, such as UV-NIL-resists.
In one embodiment said ink-pad surface is made from a material
selected from the group comprising single-crystalline materials and
polycrystalline materials, such as silicon, silicon oxide, layered
composite systems, such as silicon oxide on silicon, metal layers
on silicon/silicon oxide; amorphous materials, such as glass;
plastics, such as elastomers, preferably polydimethylsiloxane,
plastomers, preferably polyolefines (POP, polyolefinic plastomers),
ionomers, resist materials, such as UV-NIL-resists.
Preferably, said molecules to be patterned are selected from the
group comprising protein molecules, such as redox proteins,
nucleic-acid binding proteins, enzymes, metallo-proteins, such as
cytochrome c, azurin, cytoskeleton-proteins, antibodies, nucleic
acids, such as DNA, RNA, PNA, lipids, such as phospholipids and
sphingolipids.
In one embodiment said molecules to be patterned are protein
molecules having one or several lysine residues, and wherein said
substrate is Au, preferably having a spacer layer on its surface so
as to avoid denaturation of said protein, said spacer layer
preferably having a thickness in the range of from 0.5 nm to 200
nm.
In one embodiment the pattern comprises features having a length in
the range of from approximately 10 nm to 500 .mu.m, preferably
approximately 10 nm to .ltoreq.200 nm, more preferably
approximately 10 nm to .ltoreq.150 nm. It is clear that the size of
the actual features printed by the method according to the present
invention depends on the intended application of the pattern thus
printed. For example, if the intended application lies in the field
of nucleic acid chips or sensor applications, the average size of
the printed features is likely to be in the range of from 1 .mu.m
to 500 .mu.m. If the intended application lies in the field of
molecular electronics, the average size of the printed features is
likely to be in the range of from approximately 10 nm to
.ltoreq.200 nm, preferably approximately 10 nm to .ltoreq.150
nm.
The objects of the present invention are also solved by a substrate
produced by the method according to the present invention and
comprising a pattern of molecules thereon which molecules retain
their function and/or activity and/or native conformation.
The objects of the present invention are also solved by use of a
substrate according to the present invention in a sensor, a
bioreactor or for guiding cell growth.
The objects of the present invention are also solved by a device
for performing the method according to the present invention,
comprising a first means holding a solution of molecules to be
patterned, a patterned surface, preferably in the form of a stamp,
a substrate, kept in a solvent or covered by a solvent, a second
means to transfer said molecules to be patterend from said first
means to said patterned surface, a third means to transfer said
molecules to be patterned from said patterned surface to said
substrate, a fourth means to ensure that said molecules to be
patterned are kept in a solvent or are covered by a solvent during
transfer from said first means to said patterned surface to said
substrate. In a preferred embodiment of the device according to the
present invention, the second means is an ink-pad, preferably a
non-patterned surface.
The inventors have surprisingly found that it is possible to
perform a micro-contact printing process using molecules,
preferably biological macromolecules, such as proteins, nucleic
acids and/or lipids, and keeping these biological macromolecules in
solution or under solvent, preferably aqueous solvent all the time.
The method according to the present invention can be performed
using various schemes outlined further below.
As used herein, the term "molecules" is meant to denote any
molecule which may have a biological relevance. It includes nucleic
acids, including oligonucleotides, proteins, including peptides,
and lipids. The molecules may be of synthetic or natural origin. In
the case of proteins or nucleic acids, they may have sequences
occurring in nature or they may have artificial sequences.
Hence the present inventors describe an in-situ stamping process
that prevents drying or denaturation of the molecules, e.g.
proteins on the stamp after the inking process. In this .mu.CP
process, the stamp, the inkpad (if present) and the substrate are
kept during all process steps in a solvent environment, e.g. a
buffer solution, or at least covered by a buffer solution. Thus all
steps can be performed under in-situ physiological conditions.
In one aspect, the inventive method can be described by various
processes which are explained further as follows:
Process 1 (see Scheme 1 of FIG. 4):
An ink-pad is immersed in the solution of the desired molecules.
After several hours a stamp is immediately brought into contact
with the ink-pad for a few minutes in the same container. The stamp
is then rapidly transferred into a container with pure buffer
solution, so that the stamp's surface does not dry. This buffer
solution contains the substrate, on which the molecules should be
transferred. The stamp is brought into contact with the substrate
for a few minutes. Finally the modified substrate is inserted into
a buffer solution, that is free of molecules to be printed/stamped,
for storage.
Process 2 (see Scheme 2 of FIG. 4):
An ink-pad is immersed in the solution of the desired molecules.
After several hours the ink-pad is rapidly transferred into a
container with pure buffer solution, so that the ink-pad surface
does not dry. A stamp is immediately brought into contact with the
ink-pad for a few minutes. The stamp is than rapidly transferred
into another container with pure buffer solution, which contains
the target substrate. The stamp is pressed onto the substrate for a
few minutes. Finally the modified substrate is inserted into a
buffer solution for storage, that is free of molecules to be
printed/stamped.
Process 3 (see Scheme 3):
A stamp is immersed in the solution of the desired molecules. The
molecules adsorb to the stamp surface. After several hours the
stamp is rapidly transferred into a container with pure buffer
solution, so that the stamp's surface does not dry. This buffer
solution contains the substrate, on which molecules should be
transferred. The stamp is brought into contact with the substrate
for a few minutes. Finally the modified substrate is inserted into
a protein free buffer solution for storage, that is free of
molecules to be printed/stamped.
Process 4 (see Scheme 4):
The stamp of schemes 1, 2 or 3, onto which molecules to be stamped
have been immobilized is brought into contact with a substrate
under ambient conditions directly after removing it from the
ink-solution. This has to be done, as long the stamp is wet. The
stamp with the attached substrate is put immediately into a
container with pure buffer solution. After a few minutes the
transfer is finished. Finally the modified substrate is inserted
into a buffer solution for storage, that is free of molecules to be
printed/stamped.
In the following reference is made to the figures wherein the
figures show the following:
FIG. 1: SEM image of cyt c on (MUA)/gold. The lines are 1 .mu.m to
150 nm with equal gaps in between. The dark lines are cyt c
molecules.
FIG. 2: Cyclic Voltammograms (scan rate: 50 mV/s; reference
electrode: SCE) of cyt c on MUA/gold. Reference substrate without
cyt c (-), cyt c absorbed from solution (.tangle-solidup.), cyt c
after in-situ stamping or printing (.box-solid.), and cyt c after
ambient stamping or printing (.cndot.).
FIG. 3: Cyclic Voltammograms (scan rate: 50 mV/s; reference
electrode: SCE) of cyt c on MUA/gold. Comparison of different times
for which the stamp remains in a buffer reservoir before contacting
the substrate, namely 5 s (.box-solid.), 10 min (.tangle-solidup.)
and 2 h (-).
FIG. 4: shows a schematic representation of the various schemes 1-4
that are specific embodiments of the present invention.
More specifically, FIG. 4 and the schemes shown therein can be
summarized as follows:
Scheme 1:
Schematic presentation of the in-situ micro-contact printing
process. An inkpad is put into a buffered solution of Cytochrome c
for 2 h. The stamp is placed on the inkpad for 2 min. The stamp is
removed and rapidly brought into a buffer solution without drying.
Immersed into the buffer solution is a gold substrate covered with
a mercaptoundecanoic acid SAM. The stamp is brought into conformal
contact with the substrate for 2 min and is released.
Scheme 2:
Schematic presentation of the in-situ micro-contact printing
process. An inkpad is immersed into the solution containing the
desired molecules. After several hours the ink-pad is rapidly
transferred into a container with buffer solution. A stamp is
immediately brought into contact with the ink-pad for a few
minutes. Subsequently the stamp is transferred into a container
with buffer solution, which contains also the target substrate. The
stamp is pressed onto the substrate for a few minutes and is
subsequently released.
Scheme 3:
Schematic presentation of the in-situ micro-contact printing
process. A stamp is immersed in the solution of the desired
molecules. The molecules adsorb to the stamp surface. After several
hours the stamp is rapidly transferred into a container with pure
buffer solution containing the target substrate. The stamp is
brought into conformal contact with the substrate for a few minutes
and is subsequently released.
Scheme 4:
The wet stamp prepared according to Scheme 1, 2 or 3 is brought
into contact with a substrate under ambient conditions directly
after removing it from the ink-solution. The stamp with the
attached substrate is immediately immersed into a container with
pure buffer solution. After a few minutes the stamp is released
from the substrate.
Furthermore, reference is made to the following examples which are
given to illustrate, not to limit the invention.
Example
A) Stamps and Functional/Structural Investigations
It is clear to someone skilled in the art that the choice of
substrate and stamp depends on the molecules to be patterned. For
example it is clear to someone skilled in the art that for printing
nucleic acids, hydrophilic and hydrophobic substrates are suitable.
Depending on the substrate hydrophilicity the DNA may be
immobilized in from of bundles (hydrophobic surface) or as
individual strands (hydrophilic substrate). It is also clear to
someone skilled in the art that for the transfer of proteins
containing cystein groups onto gold a spacerlayer (e.g
Mercapto-SAM) has to be used to cover the bare gold surface in
order to prevent the binding of cystein to gold which may denature
the protein. On the other hand it is clear to someone skilled in
the art that a hydrophilic polypeptide (with undefined tertiary and
quaternary structure) like polylysin can be printed to a
hydrophilic siliconoxide surface. It is also clear to someone
skilled in the art that the choice of the stamp material depends on
the pattern size. The minimum pattern size is strongly dependent on
the tensile modulus of the materials, e.g PDMS with a tensile
modulus of 1 MPa is good for printing patterns down to 300 nm,
while for patterns below 300 nm Polyolefine with a tensile modulus
of 1 GPa may be suitable. It is also clear to someone skilled in
the art that for printing patterns on large areas a flexible stamp
made out of a flexible plastic material is more preferable than
made of any other material, because the flexible stamp is able to
make a conformal contact on the whole area. It is also clear to
someone skilled in the art that the hydrophilicity of the stamp and
the substrate and the solubility of the biomolecules to be
transferred determine the interaction of the bio-molecules with the
inkpad, the stamp and the substrate.
The stamps for the process can generally be made from elastomers,
plastomers, ionomers, resist materials, and also from hard
materials such as crystalline and polycrystalline materials. It is
also possible to use a combination of these materials for the
preparation of composite stamps (soft-soft, soft-hard, and
hard-hard). The stamps are prepared either by drop casting and
thermal- or photo-induced curing or by hot embossing techniques
from masters that are, if required, passivated with a release
layer, e.g. a monolayer of
(1,1,2,2,-tridecafluoro-octyl)-trichlorosilane, or sodium dodecyl
sulfate (SDS). The surface of the stamp surface can be chemically
modified by exposing the stamp surface to e.g. an oxygen plasma or
by chemically reacting it.
For the specifically disclosed embodiments further below, POP
(polyolefine plastomer) (Affinity VP 8770G) from Dow Chemicals was
used. POP was heated up to 85.degree. C. and pressed with 90 kPa
into a silicon oxide (Sioxide) master, which is passivated with
monolayer of (1,1,2,2,-tridecafluoro-octyl)-trichlorosilane (Sigma
Aldrich).
All specific embodiments were performed using horse heart
cytochrome c as a model system.
The redox activity of the proteins was investigated using a PAR
Model 283 potentiostat controlled by a PC running version 2.4 of
CorrWare software. The working electrode was an Au(111) single
crystal cylinder with a diameter of 3.5 mm. For the measurement the
hanging meniscus method was applied. In this method the particular
metal plane of the single crystal is brought into contact with the
electrolyte by forming a meniscus. The Au crystal was deaned in
sulfuric acid. After flame annealing the Au(111) crystals was
placed for 10 min into mercaptoundecanoic acid (MUA)
(Sigma-Aldrich) and subsequently rinsed with ethanol and MilliQ
water (18.2 M.OMEGA., total amount of carbon 3-4 ppm). A standard
calomel electrode (SCE) was used as reference electrode; the
counter electrode is a platinum coil. The setup was placed in a
Faraday cage to reduce electronic noise.
For SEM imaging, a 5 nm chromium and 50 nm gold layer was
evaporated onto a piece of silicon oxide wafer. The chip was also
cleaned with sulfuric acid, flame annealed and placed into a 10 mM
ethanolic solution of MUA (Sigma-Aldrich) for 10 min.
B) Preparation and Printing
A sodium phosphate solution (Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4)
(Merck) at pH 7 with a concentration of 3.26 mM was used as a
buffer to prepare a 12.6 .mu.M hourse heart cyt c (Sigma-Aldrich)
solution. A Polydimethylsiloxane (PDMS) (Sylgard 184, Dow Coming)
inkpad was immersed into the cyt c solution. After 2 h, the stamp
was introduced into the solution and gently pressed onto the inkpad
for 2 min. Immediately after the separation of the inkpad and the
stamp, the stamp was introduced into the buffer solution containing
the substrate without drying, in order to cover the proteins with a
thin wet film maintaining the desired physiological conditions
during the transfer of the stamp from one solution to the other.
The transfer time was less than 5 s. After transferring the stamp
into the solution containing the substrate, the stamp was brought
into conformal contact with the substrate for 2 min by applying
gentle finger pressure, for the transfer of the proteins from the
stamp onto the MUA modified Au substrate. The immobilization of cyt
c onto a MUA SAM is based on electrostatic interaction.
The acid group of MUA is deprotonated and thus negatively charged,
while cyt c has a positive net charge. Since the positive charge of
the lysine groups are located on one side of the cyt c, the
orientation of cyt c on the surface is always the same. FIG. 1
shows a SEM image of the transferred pattern. The four 150 nm broad
lines are dearly separated by a 150 nm wide gap (right side). The
interspacing between transferred lines is free of molecules. No
common drawbacks or shortcoming of .mu.CP like sagging or diffusion
of the ink molecules can be seen.
C) Cyclic Voltammetry Measurements
The redox activity of proteins stamped with an unstructured stamp
onto a Au/Cr coated Si/Sioxide wafer was measured with cyclic
voltammetry. The proteins were stamped in the same way as described
under B). After transferring the proteins onto the substrate, the
substrate was directly transferred into the measurement cell. FIG.
2 shows cyclic voltammograms of cyt c after the in-situ .mu.CP
process ("in-situ printing") according to the present invention in
comparison with cyt c stamped under ambient conditions, i.e. where
cyt c was dried on the stamp ("printing under ambient conditions)
and a voltammogramm of cyt c adsorbed from solution ("cyt c in
solution"). In all cases a distinct and reversible redox peak
occurred at a redox potential of E.sup.0=-60 mV. The symmetry of
the peak indicates that cyt c is adsorbed onto a surface. The
current at the redox potential is comparable for the samples
prepared by in-situ printing and by adsorption from solution, while
the current observed for the proteins transferred under ambient
conditions is 70% smaller. This could be either due to a lower
protein density or result from a partial loss in functionality due
to the drying process.
One, important aspect for the process appears to be the time
duration the stamp is exposed to the buffer solution without being
in conformal contact with the substrate. FIG. 3 shows redox
activity of transferred proteins after the stamp was exposed to
buffer solution for 5 s to 120 min(5 s, 10 min and 2 h) prior to
the transfer process. The longer the stamp was exposed to the
buffer solution, the lower was the current at the redox potential.
The reduction in the current is due to a decrease in the surface
coverage on the stamp. Since the proteins are only weakly adsorbed
by London forces to the stamp surface, the concentration gradient
drives desorption of the proteins into the buffer solution.
The process according to the present invention allows to pattern
biomolecules to dimensions down to 150 nm, while preserving their
structural integrity and functionality by using an in-situ
process.
The features of the present invention disclosed in the
specification, the claims and/or in the accompanying drawings, may,
both separately, and in any combination thereof, be material for
realizing the invention in various forms thereof.
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